Glucose is the major source of energy in cells. Bacteria typically use two metabolic pathways to catabolize glucose: the glycolysis and, to a lesser extent, the pentose phosphate pathway. Glycolysis converts glucose into pyruvate, and the free energy released is used to form ATP (adenosine triphosphate) and NADH (reduced nicotinamide adenine dinucleotide). The pentose phosphate pathway utilizes glucose to generate various substrates, and produces NADPH (reduced nicotinamide adenine dinucleotide phosphate) and pentoses. More specifically, the pentose phosphate pathway generates NADPH from NADP, in conjunction with the reduction of glucose-6-phosphate into ribulose-5-phosphate.
Although bacteria utilize mainly glycolysis to catabolize glucose, NADPH, which is produced during the pentose phosphate pathway, has a key role in biological reactions that lead to industrially useful compounds. Indeed, cells use NADPH as reducing equivalents for many biosynthetic and oxidation-reduction reactions involved in the protection against the toxicity of reactive oxygen species (ROS), allowing the regeneration of reduced glutathione. NADPH is also used in several important anabolic pathways, such as amino acid synthesis (e.g., arginine, proline, isoleucine, methionine, lysine), vitamin synthesis (e.g., pantothenate, phylloquinone, tocopherol), polyol synthesis (e.g., xylitol), isoprenoid synthesis, and fatty acid synthesis (including polyunsaturated fatty acids), as well as in the synthesis of other high added-value substances. In addition, NADPH is the source of reducing equivalents for cytochrome P450 hydroxylation of aromatic compounds, steroids, alcohols and drugs.
Because of the involvement of NADPH in the capacity of cells to conduct important enzymatic reactions, recombinant approaches have been proposed in the art to provide microorganisms with increased cellular NADPH.
For instance, attempts have been made to limit the activity of enzymes involved in the oxidation of NADPH, and/or to increase the activity of enzymes involved in the reduction of NADP. For example, U.S. patent application Ser. No. 10/577,084 relates to strains of microorganisms having one or more of their NADPH-oxidizing activities limited by a deletion of at least one gene coding for a quinine oxidoreductase or a soluble transhydrogenase.
U.S. Pat. No. 5,830,716 relates to a recombinant Escherichia coli expressing a nicotinamide dinucleotide transhydrogenase. In this bacterium, increased NADPH levels are produced from NADH.
Such approaches allow the production of modified microorganisms having an increased NADPH/NADP ratio. However, modifying the cell metabolism by deregulating specific genes is not entirely satisfactory. Indeed, the metabolic flux in such cells is not stable and cannot be controlled. Furthermore, the metabolic flux may not be adapted easily because the activity of such recombinant cofactor regeneration systems is strictly controlled by and dependent on the robustness of the bacteria physiology. In addition, the production of such recombinantly engineered organisms is costly and time-consuming, with repercussions on the production of the compounds of interest.
The over-expression of glucose-6-phosphate dehydrogenase to increase NADPH production has also been considered. However, over-expression of the corresponding coding gene is lethal for the cells.
There is therefore always a need for microorganisms suitable for producing industrially useful compounds. There is also a need for a source of reductive or anti-oxidant activity. There is also a need for improved methods for generating reduced compounds or anti-oxidant compositions.